1. Field of the Invention
The invention encompasses antikine antibodies, or antibodies that bind to two, three, four, five, or more CC chemokines (CC chemokines are also known as β-chemokines), particularly those antibodies which bind to at least two chemokines selected from the group consisting of RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, and MCP-1/CCL2. Unlike antibodies that bind to only a single CC chemokine, antikine antibodies practically address the problem of functional redundancy amongst CC chemokines by binding to, detecting, and/or neutralizing more than one CC chemokine at a time. Other aspects of the invention include diagnostic and therapeutic uses of antikine antibodies including the treatment of conditions, disorders, or diseases mediated by CC chemokines; hybridoma cell lines producing antikine antibodies and methods for producing hybridomas by sequential immunization; methods for humanizing antikine antibodies; and methods for improving antikine antibodies by affinity maturation.
2. Description of the Related Art
Chemokines are key mediators of inflammation and are implicated in the development of autoimmune disease; Viola & Luster, Ann. Rev. Pharmacol. Toxicol. 48: 171-197 (2008). They are produced at sites of inflammation or infection and induce the migration of leukocytes from the circulation into the tissue. Simple and effective ways for modulating inflammation or immunological processes mediated by chemokines have long been sought. These efforts are complicated by the redundancy and overlap in the functions of numerous different chemokines and their receptors. For example, chemokine inhibitors have been used to treat autoimmune conditions in preclinical animal models, but have yet to succeed in the clinic for treating autoimmune indications. It has been proposed that this lack of efficacy may be due to the redundancy in the functions of chemokines. Over 50 different chemokines have been identified and each has different structural and functional properties. The specificity of some chemokines overlap, that is, they bind to the same type of receptor or act on similar types of cells; Vergunst, et al., Arthritis Rheum. 58: 1931-1939 (2008). A particular chemokine may bind to more than one type of chemokine receptor and a given chemokine receptor may be bound by more than one type of chemokine. Therefore, the development of a single agent capable of binding to, blocking chemokine binding to a receptor, or otherwise neutralizing the activity of more than one chemokine is highly desirable.
Chemokines which take their name from chemotactic cytokines, are small secreted polypeptides that regulate movement of immune cells into tissues; Baggiolini, et al., Adv. Immunol. 55:97-179 (1994); Oppenheim et al., Ann. Rev. Immunol. 9:617-648 (1991).
All chemokines share a Greek key structure that is stabilized by disulfide bonds between conserved cysteine residues. However, chemokines are further assigned to four different families based on the number and position of these conserved cysteine residues. The α- and β-chemokines each contain four conserved cysteine residues. The first two cysteines of an α-chemokine are separated by a single amino acid, thus forming a characteristic CXC amino acid motif. The first two conserved cysteines of a β-chemokine are adjacent. Thus, the β-chemokines are also known as CC chemokines. By contrast, lymphotactin is the sole member of a third class of XC chemokines and contains only the second and fourth conserved cysteine residues. A fourth class of chemokines, of which fractalkine is the sole member, is the CXXXC or CX3C class which has 3 amino acids separating the first two conserved cysteines. In humans, α-chemokines are mainly encoded by genes clustered on chromosome 4 and β-chemokines are mainly encoded by genes on chromosome 17. Lymphotactin is encoded on chromosome 1 and fractalkine on chromosome 16.
Chemokines form gradients that serve as chemoattractants and potential proliferation signals for immune and other cells such as monocytes, macrophages, basophils, eosinophils, T lymphocytes and fibroblasts. CC chemokines exhibit chemoattractant properties by forming concentration gradients recognized by chemotactic cells; CC chemokines also signal particular cell types to proliferate, including fibroblasts and immune cells such as monocytes, macrophages, T lymphocytes, basophils, and eosinophils. The target receptors and cells of chemokines, including CC chemokines are described by Viola, et al., Annu. Rev. Pharmacol. Toxicol. 48:171-197 (2008), see e.g., FIG. 1, which document is hereby incorporated by reference for its teachings regarding CC chemokine chemoattractant and signaling functions.
Chemokines share structural features associated with particular chemokine functions, such as with binding to a chemokine receptor. Common structures include an elongated N-terminus segment (N terminal domain) that precedes the first cysteine residue, N loop, 310 helix, beta strands β1, β2, and β3, the 30's, 40's and 50s loops; location of disulfide bonds, and the C-terminal α-helical segment.
These and other CC chemokine structures, including conserved or homologous amino acid residues among different CC chemokines, as well as the solvent-accessible, partially solvent accessible, and buried amino acid residues of CC chemokines are incorporated by reference Fernandez, et al., Annu. Rev. Pharmacol. Toxicol. 42:469-99 (2002), see e.g., FIGS. 1 and 2. Buried amino acid residues of intact, undenatured chemokines are unlikely to form epitopes or antigenic determinants contacted by antibodies to a chemokine. In contrast, solvent or surface-exposed CC chemokine residues are more accessible to antibody binding.
CC chemokine residues associated with chemokine receptor binding of CCL3/MIP-1α include residues 11-15 (CCFSY), residues 17-24 (SRQIPQNF), residues 34-35 (QC), and residues 57-67 (EWVQKYVSDLE) of SEQ ID NO: 71;
residues associated with CCL4/MIP-1β chemokine receptor binding include residues 11-15 (CCFSY), residues 17-24 (ARKLPHNF), residues 34-35 (LC), or residues 57-67 (SWVQEYVYDLE) of SEQ ID NO: 72;
residues associated with CCL5/RANTES binding include residues 10-14 (CCFAY), residues 16-23 (ARPLPRAH), residues 33-34 (KC), or residues 56-66 (KWVREYINSLE) of SEQ ID NO: 73; residues associated with CCL23/MPIF-1 binding include residues 9-13 (CCISY), residues 15-22 (PRSIPCSL), residues 32-33 (EC), or residues 55-65 (KQVQVCMRMLK) of SEQ ID NO: 81; and
residues associated with CCL15/HCC-2 include residues 8-12 (CCTSY), residues 14-21 (SQSIPCSL), residues 31-32 (EC), or residues 54-64 (PGVQDCMKKLK) of SEQ ID NO: 79. Corresponding amino acid residues of other CC chemokines are depicted, for example, by FIG. 1 of Fernandez, et al., id. (2002).
Conserved domains for CC chemokines are disclosed at http://www.ncbi.nlm.nih.gov/. This structural data is incorporated by reference to the protein and conserved domain database information at the website named above as last accessed Aug. 24, 2010.
Chemokines in the CC chemokine class interact with seven transmembrane G-protein coupled receptors termed CC chemokine receptors or CCRs, Rossi & Zlotnik, Ann. Rev. Immunol. 18:217-242 (2002). Interaction of the chemokine with its receptor regulates activation of adhesion molecules and affects diapedesis and extravasation of immune cells from the circulation into tissues.
Chemokines have been implicated in the development and maintenance of numerous inflammatory and immunological conditions, disorders and diseases. These include rheumatoid arthritis, multiple sclerosis, atherosclerosis, psoriasis, inflammatory bowel disease (including Crohn's disease, ulcerative colitis, Celiac disease), vascular restenosis, lupus nephritis, glomerulonephritis, transplant rejection, scleroderma, fibrotic disease, asthma (and other lung inflammatory conditions). For example, levels of CC-chemokines are elevated in affected tissues from patients with rheumatoid arthritis, multiple sclerosis (MS), atherosclerosis, and others. Preclinical animal models of these diseases show that inhibition of individual chemokines can at least partially ameliorate disease symptoms. For example, Kasama, et al., J. Clin. Invest. 95: 2868-2876 (1995) demonstrated that administration of an antibody which inhibited MIP-1α/CCL3 could reduce arthritis clinical scores by approximately 50% in a rodent model of rheumatoid arthritis. Similarly, Ogata, et al., J. Pathol. 182: 106-114 (1997) showed that an anti-MCP-1/CCL2 antibody could decrease joint swelling by approximately 30%. Receptors for MIP-1α/CCL3 (including CCR1 and CCR5) and MCP-1/CCL2 (CCR2) are expressed in an overlapping pattern in leukocytes, and thus it is possible that an inhibitor of both MIP-1α/CCL3 and MCP-1/CCL2 could be more efficacious than individual inhibitors of each chemokine alone. Viola, et al., Annu. Rev. Pharmacol. Toxicol. 48:171-197 (2008), is hereby incorporated by reference for its disclosure of specific classes or types of diseases and disorders associated with or mediated by such CC chemokines, see e.g., Table 1.
Natural inhibitors of chemokine activity are known and specific agents, such as antibodies or small molecule inhibitors that bind to or interfere with the activity of particular chemokines have been developed, see Fernandez, et al., Annu. Rev. Pharmacol. Toxicol. 42: 469-99 (2002), to which such inhibitors and agents are incorporated by reference, see e.g., pages 482-488. Vaccinia and related pox viruses produce a soluble 35 kD protein termed vCCI (SEQ ID NO: 117) which binds and inhibits multiple chemokines within the CC class of chemokines. The CC class of chemokines generally acts upon leukocytes including T cells and monocytes; Smith et al., Virology 236: 316-327 (1997), Burns et al., J. Biol. Chem. 277: 2785-2789 (2002). Recombinant vCCI has been shown to be effective in reducing leukocyte infiltration in several models of chronic inflammatory disease, including experimental autoimmune encephalitis; Jones et al., Cytokine 43: 220-228 (2008) and asthma; Dabbagh, et al., J. Immunol. 165: 3418-3422 (2000). However, use of natural substances such as viral proteins like vCCI foreign to a subject's immune system raises safety issues. Administration of substances, such as vCCI can induce undesired physiological or immune responses and such substances can be neutralized, removed or destroyed as foreign by host clearance or defense mechanisms.
With this in mind, the inventors focused on developing a method for producing antibodies, especially humanized antibodies, that can specifically bind to and neutralize more than one chemokine, but which do not pose the risks associated with molecules like vCCI. Prior art chemokine inhibitors, such as antibodies that bind to a single CC chemokine suffer from the problem of CC chemokine receptor redundancy. For example, CCL3/MIP-1α and CCL5/RANTES each bind to chemokine receptors 1 (CCR1) and 5 (CCR5), see FIG. 1 of Viola, id. (2008). An antibody that only inhibits CCL3 binding to these chemokine receptors wouldn't prevent receptor activation by the binding of other CC chemokines, such as CCL5.
The inventors initially targeted CC chemokines MIP-1α/CCL3, MIP-1β/CCL4 and RANTES/CCL5, which constitute the primary ligands for chemokine receptors CCR1 and CCR5. CCL2/MCP-1 was also targeted as a ligand for CCR2. As shown by FIG. 1 of Viola, et al., id. (2008), these receptors are broadly expressed on monocytes and T cells, as well as on other leukocyte subsets. They are implicated in numerous inflammatory disease states both in preclinical animal models of disease and in human disease.